This application incorporates by reference in its entirety the Sequence Listing that is provided in duplicate on compact discs that accompany the application. Each CD contains the following file: 1020C2, having a date of creation of Mar. 18, 2002 and a file size of 2.90 MB.
This invention relates to the field of modifying the responses of plant cells to external signals, such as environmental changes, and developmental cues. More specifically, this invention provides isolated polynucleotides encoding polypeptides that are integrally located in plant cell membranes and that mediate cellular signaling processes.
Plants progress through set developmental programs throughout the course of their lifetimes. This is particularly evident in embryogenesis and floral development. There are a variety of signal molecules produced by certain cells in the plant to which other cells, particularly in the meristematic regions, arc poised to respond. These signal molecules trigger distinct sets of developmental programs at specific times that lead to the formation of, for example, flowers or cotyledons. In addition to the programmed developmental pathways, plants are exposed to a variety of environmental stimuli such as changes in temperature and amount of sunlight, availability of water, wounding from mechanical injury and attack by pathogens. Environmental factors, such as exposure to light, heat, cold, drought, etc., activate the expression of genes and synthesis of proteins and other compounds essential for an appropriate response to the environmental signal and thereby, the healthy development of the plant. These responses, like the developmental pathways, are mediated by signal molecules (hereinafter referred to as ligands).
To respond to these ligands, plant cells produce surface receptor proteins that serve as sensors, regulators; and/or transducers of cell signals. The intracellular transduction of a signal is often transmitted via a phosphorylation cascade of molecules that culminates in the transcription of genes to elicit the appropriate cellular response either for normal development or against environmental challenge.
One major class of receptor proteins is the single-transmembrane family, of which there are several subclasses. These proteins are characterized by three domains: an extraccllular signal molecule recognition/binding domain, a single cell membrane-spanning domain and an intracellular signal transduction domain which is usually a protein kinase. Many, but not all, plant single transmembrane proteins belong to the subclass known as receptor-like kinases (RLKs). The intracellular kinase domains of plant RLKs are all serine/threonine protein kinases, while the extracellular domains of RLKs are of different types. One type of RLK is characterized by the presence of the extracellular S-domain, originally described in self-incompatibility-locus glycoproteins that inhibit self-pollination. The S-domain is recognized by an array of ten cysteine residues in combination with other conserved residues. Another class of RLKs has an extracellular domain distinguished by leucine rich repeats (LRR) that are involved in protein-protein interactions. Binding of ligands to the extracellular domain is followed by receptor dimerization, autophosphorylation and the activation of a series of intracellular proteins which serve to transduce the signal to the nucleus. The structure of plant RLKs is very similar to receptors found in cell signaling pathways in animal systems.
One example of a plant RLK is the Xa21 gene, which confers resistance to the plant pathogen Xanthomonas oryzae pv. oryzae race 6. This gene was cloned using genetic means comparing Xanthomonas-sensitive and resistant strains of rice (Song et al., Science 270:1804-1806, 1995), and has been subsequently shown to confer resistance to Xanthomonas in Arabidopsis. The 1025 amino acid protein shows a number of features with similarity to known protein domains including a NH2-terminal 23 amino acid residue signal peptide, indicating that the protein is directed to the plasma membrane. Amino acids 81 to 634 contain 23 imperfect copies of a 24-amino acid LRR. Amino acids 651 to 676 encode a 26-amino acid hydrophobic segment that is likely to form a membrane-spanning domain. The C-terminal amino acids contain a putative intracellular serine threonine kinase domain carrying 11 subdomains with all 15 invariant amino acids that are typical of protein kinases. Subdomains VI and VIII are indicative of serine-threonine phosphorylation specificity. Xa21 has strong similarities to other RLKs, such as the Arabidopsis receptor-like kinase proteins RLK5 (HAESA) and TMK1, showing conservation of both the LRR and protein kinase domains. It is not yet known to what protein Xa21 transduces its pathogen recognition signal.
Another family of membrane receptor molecules expressed by plant cells is histidine kinases (HKs). HKs have been known for some time in bacterial signal transduction systems, where they form one half of a two-component signaling system. The bacterial HK serves as a sensor molecule for extracellular signals, such as changes in osmoticum, nutrients and toxins. The HK autophosphorylates on a histidine residue in response to ligand binding. This phosphohistidine donates its phosphate group to an aspartate residue of the second member of the two component system, known as the response regulator (RR). The phosphorylated RR then goes on to further transduce the signal, by binding other proteins as regulatory subunits, thereby either activating or inactivating them, depending on the specific circumstance. Alternatively, the phosphorylated RR binds DNA in a sequence-specific manner, serving to directly activate specific genes which code for proteins that mediate the response to the extracellular stimulus. In certain cases, HKs have a composite structure. Specifically, these proteins contain RR domains at their carboxy termini. The phosphohistidine of the HK transfers its phosphoryl group to the active site aspartate residue of this RR domain. In these cases, since the RR domain is membrane-bound, the signal cannot be transduced directly by RR binding to DNA. Instead, histidine phosphotransfer (HPt) proteins serve to further transduce the signal. The phosphoaspartate of the composite HK/RR protein donates the phosphate group to an active site histidine in the HPt protein. The HPt phosphohistidine in turn donates the phosphate group to a true RR, which then modulates activities of other proteins or activates gene expression in response to the external signal.
Like bacteria, plant cells have several two-component signaling systems which consist of a sensor element HK and a RR. In addition, composite HK proteins with RR domains at their carboxy termini (hereinafter referred to as hybrid HK/RR proteins) are found in both bacteria and plants. The HK proteins are distinguished by well-conserved amino acid motifs that occur in a specific order. From the amino terminus, the conserved regions are identified as the H, N, G1, F and G2 boxes. These motifs are usually found within a 200-250 amino acid span of the protein. The G1, F and G2 boxes are thought to be involved in nucleotide binding. As in bacteria, upon receiving the extracellular ligand, the HK is autophosphorylated on the histidine residue contained in the H box. The phosphate group is subsequently transferred to the RR. Alternatively, some HKs constitutively autophosphorylate their histidine residues and this activity is suppressed by binding of the extracellular ligand. All HKs are believed to phosphorylate a RR, as an obligate part of signal transduction.
RRs are characterized by the absolute conservation of an aspartate which is phosphorylated by the phosphohistidine of the HK, and a conserved lysine residue. Unlike bacteria, RRs in plants have not been shown to bind DNA directly. Rather, all the plant RR""s characterized to date appear to transduce the signal into protein kinase cascades, which eventually phosphorylate and either activate or inactivate transcription factors, and thereby gene expression. Similar to bacteria, plants also contain hybrid HK/RRs which contain a RR domain at the carboxy terminus of the protein. As might be expected based on this observation, plant genomes have also been found to harbor histidine-containing phosphotransfer (HPt) domain genes. The HPt domain has been shown to play an important role in some His-Asp phosphorelay pathways. However, it has not yet been shown directly that any plant HPt protein interacts either with a hybrid HK/RR or with a soluble RR.
The ethylene receptor family (e.g., ETR1; Chang et al., Science 262:539-544, 1993) comprise the best known two-component signaling system in plants. Ethylene is a well-known ligand that is involved in the coordination of fertilization, senescence, skoto/photomorphogenesis, and responses to pathogens and mechanical injury. The ethylene signal is transduced through the protein CTR1, which is a Raf-like protein kinase. CTR1 is a negative regulator of downstream steps in the signaling pathway. While the details of this pathway remain unclear, it appears that the ethylene receptors are constitutively active in the absence of ethylene, thereby constantly phosphorylating CTR1, which in turn represses other genes in the ethylene response pathway. Binding of ethylene to the ethylene receptors inhibits the phosphorylation function of the receptor, which results in the inhibition of the negative regulator CTR1, thereby allowing the activation of downstream proteins in the ethylene signal transduction cascade. This culminates in activation of ethylene response genes.
Two RR genes, IBC6 and IBC7, which are induced in response to the plant growth regulator cytokinin, have been cloned from Arabidopsis thaliana and characterized (Brandstatter and Kieber, Plant Cell 10:1009-1019, 1998). Cytokinin is known to regulate plant growth and development, including such physiological events as nutrient metabolism, expansion and senescence of leaves, and lateral branching. It is likely that IBC6 and IBC7 are involved in the transduction of the cytokinin signal in plants. Consistent with such a hypothesis, it has been demonstrated that ARR4 (IBC7) interacts directly with phytochrome B (phyB) (Sweere et al., Science 294:1108-1111). PhyB is one of a family of histidine kinase-like photoreceptor molecules (Thummler et al., FEBS Lett. 357:149-155, 1995; Kehoe and Grossman, Science 273:1409-1412, 1996) involved in photo/skotomorphogenesis and physiological responses to day length, such as initiation of flowering. The finding of IBC7""s interaction with phyB is a link between cytokinin perception and cytokinin""s physiological effects on prevention of light deprivation-mediated etiolation and senescence. Furthermore, the response regulator ARR1 has been shown to function as a transcription factor which directs the transcription of ARR6, another response regulator that is transcribed in direct response to cytokinin, like IBC6 and IBC7 (Sakai et al., Science 294:1519-1521, 2001). The proteins encoded by these RR genes are all possible signal transduction partners with the hybrid HK/RR known as CRE1/WOL. CRE1/WOL was recently shown to directly bind cytokinin and transduce a signal in transgenic yeast cells (Inoue et al., Nature 409:1060-1063, 2001; Suzuki et al., Plant Cell Physiol. 42:107-113, 2001). Subsequent studies have further shown that CRE1/WOL directly binds cytokinin in Arabidopsis thaliana (Yamada et al., Plant Cell Physiol. 42:1107-1123, 2001; Ueguchi et al., Plant Cell Physiol. 42:751-755, 2001). Furthermore, the gene encoding the hybrid HK/RR protein CKI1 causes cytokinin-like effects when it is ectopically expressed in transgenic plants (Kakimoto, Science 274:982-985, 1996). However, the role that the CKI1 protein plays in cytokinin signal transduction, if any, is still unclear. It is possible that any or all of the known plant response regulators may also interact with CKI1 to mediate other aspects of the cytokinin response. However, it is clear that a two-component HK/RR system is involved in cytokinin signal transduction.
While polynucleotides encoding proteins involved in plant cell signaling have been isolated for certain species of plants, genes encoding many such proteins have not yet been identified in a wide range of plant species. Thus, there remains a need in the art for materials which may be usefully employed in the modification of cell signaling in plants.
Briefly, the present invention provides polynucleotides isolated from eucalyptus and pine which encode polypeptides involved in cell signaling, together with methods for the use of such polynucleotides and polypeptides. Such polypeptides function as sensor-regulators or receptor kinases. The isolated polynucleotides and polypeptides may be usefully employed in the modification of plant cell responses either during the growth and development of a plant, or under conditions of stress resulting from pathogens or environmental factors.
In a first aspect, the present invention provides isolated and purified polynucleotides obtainable from eucalyptus and pine which encode RLKs, HKs, RRs, HPts or hybrid HK/RR proteins. In one embodiment, the isolated polynucleotides comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 1-67, 131-481, 833-888, 946-952 and 960-974; (b) complements of the sequences recited in SEQ ID NO: 1-67, 131-481, 833-888, 946--952 and 960-974; (c) reverse complements of the sequences recited in SEQ ID NO: 1-67, 131-481, 833-888, 946-952 and 960-974; (d) reverse sequences of the sequences recited in SEQ ID NO: 1-67, 131-481, 833-888, 946-952 and 960-974; and (e) sequences having either 75%, 90% or 95% identity, as defined herein, to a sequence of (a)-(d).
In a further aspect, isolated polypeptides encoded by an inventive polynucleotide are provided. In certain embodiments, such polypeptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 68-130, 482-832, 889-945, 953-959 and 975-989; and sequences having at least 75%, 90% or 95% identity to a sequence of SEQ ID NO: 68-130, 482-832, 889-945, 953-959 and 975-989.
In another aspect, the invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, in combination with one or more other polynucleotides disclosed herein, or in combination with one or more known DNA sequences, together with transgenic cells comprising such constructs.
In a related aspect, the present invention provides genetic constructs comprising, in the 5xe2x80x2-3xe2x80x2 direction, a gene promoter sequence; an open reading frame coding for at least a functional portion of a polypeptide of the present invention; and a gene termination sequence. The open reading frame may be orientated in either a sense or antisense direction. Genetic constructs comprising an untranslated, or non-coding, region of a gene coding for an inventive polypeptide or a nucleotide sequence complementary to a non-coding region, together with a gene promoter sequence and a gene termination sequence, are also provided. Preferably, the gene promoter and termination sequences are functional in a host plant. Most preferably, the gene promoter and termination sequences are those of the original genes but others generally used in the art, such as the Cauliflower Mosaic Virus (CaMV) promoter, with or without enhancers such as the Kozak sequence or Omega enhancer, and Agrobacterium tumefaciens nopaline synthase terminator may be usefully employed in the present invention. Tissue-specific promoters may be employed in order to target expression to one or more desired tissues. The genetic construct may further include a marker for the identification of transformed cells.
In a further aspect, transgenic cells, preferably plant cells, comprising the genetic constructs of the present invention are provided, together with organisms, preferably plants, comprising such transgenic cells, and fruit and seeds and other products, derivatives, or progeny of such plants. Propagules of such transgenic plants are also encompassed in the present invention. As used herein, the word xe2x80x9cpropagulexe2x80x9d means any part of a plant that may be used in reproduction or propagation, sexual or asexual, including cuttings
In yet another aspect, methods for modifying cell signaling in a target organism, such as a plant, are provided, such methods including stably incorporating into the genome of the plant a genetic construct of the present invention. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. In a related aspect, a method for producing a target organism, such as a plant, having modified cell signaling is provided, the method comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
In yet a further aspect, the present invention provides methods for modifying the activity of a polypeptide in a target organism, such as a plant, comprising stably incorporating a genetic construct of the present invention into the genome of the plant. In a preferred embodiment, the target plant is a woody plant, preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus grandis and Pinus radiata. 
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the drawings and the following more detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.